Two mechanisms of nucleoside transport, one sensitive to nanomolar concentrations of nitrobenzylthioinosine (NBTI), the other insensitive, will be examined in mammalian cells. NBTI-sensitive transport appears to be the major mechanism for nucleoside uptake in most mammalian cells; whereas NBTI-insensitive transport is quantitatively less important, but may play a significant role in the uptake of cytotoxic nucleosides when the major transport mechanism is blocked by NBTI. The basic properties of these two transport processes (kinetics, substrate specificity and sensitivity to inhibitors) will be examined in two model cells, S49 murine lymphoma and L1210 murine leukemia, which differ dramatically in their relative levels of NBTI-sensitive and -insensitive transport activity. Specific monoclonal antibodies to each transport process will be obtained using hybridoma technology. These antibodies will be used in combination with membrane labeling studies to identify plasma membrane proteins associated with NBTI-sensitive and -insensitive nucleoside transport activity and to distinguish whether these activities are properties of two distinct proteins or one complex nucleoside transporter. Transport activity and membrane labeling patterns will also be examined to transport deficient mutants to further define the relationship between these two processes. Nucleoside transport proteins will be isolated frm L1210 cells and reconstituted in liposomes in order to study their basic properties free of the influence of each other, and/or nucleoside metabolizing enzymes. These studies of the biochemical properties of nucleoside transport are expected to provide a firm foundation for future investigations into the role of NBTI-insensitive transport in the uptake of cytotoxic nucleosides, with the long-range goal of predicting which tumors may be susceptible to therapy through modulation of intracellular drug levels by agents which selectively modify nucleoside transport activity.